Conjugates were prepared by carbodiimide-mediated coupling of adipic acid dihydrazide derivatiaves of H. influenzae type b (Hib), pneumococcus type 6A (Pn6A) and E. coli K100 with tetanus toxoid (TT) and the immunogenicity characterized in laboratory mice and in primates. The Pn6A conjugates were less immunogenic than the Hib conjugates in laboratory animals. The immunogenicity of K100-TT was similar to the Hib-TT, but some variability was noted in both though their physical-chemical charactristics such as molecular size and polysaccharide/protein ration were similar. Studies were initiated to further characterize the conjugates by physical-chemical methods to enable in vitro prediction of immunogenicity (standardization). Preliminary results of applying the Hib-TT to agarose gel electrophoresis showed that about two-thirds of the conjugate could be electrophoresed in a 2% gel; about one-third would not enter the gel. The electrophroesis was regulated by the ionic strength of the buffer used; low ionic buffer would aggregate the conjugate. In search for methods of preparing conjugates reproducibly, a method CNBr activation at neutral pH was adjusted to Hib, and sonication was utilized to produce polysaccharides of similar, lower molecular size. Attempts at reproducibly derivatizing E. coli K1 polysaccharide were not successful. Adherence to mucous membranes is an important initial event in bacterial colonization and may be related to their pathogenicity. Pili, shown to be the adherence mechanism in several bacterial species, have been recently demonstrated in H. influenzae. Studies were initiated to isolate and purify Hib pili, to evaluate their role in pathogenesis and immunity. Pili expression of several H. influenzae type b strains from disease isolates; CSF and epiglotitis and from carriers, was enriched utilizing their adherence properties to human RBC.